The present invention relates generally to systems and methods for analyzing the reactive sites of substrates, in particular drugs. More specifically, the invention relates to systems and methods for determining the relative metabolic reaction rates of these reactive sites, especially with respect to alternative reaction pathways in the cytochrome p450 enzymes, so as to model and predict the metabolic properties of the substrate, as well as to design and redesign such substrates in order to achieve desired metabolic properties.
Drug development is an extremely expensive and lengthy process. The cost of bringing a single drug to market is about $500 million to $1 billion dollars, with the development time being about 8 to 15 years. Drug development typically involves the identification of 1000 to 100,000 candidate compounds distributed across several compound classes that eventually lead, to a single or several marketable drugs.
Those thousands of candidate compounds are screened against biochemical targets to assess whether they have the pharmacological properties that the researchers are seeking. This screening process leads to a much smaller number of xe2x80x9chitsxe2x80x9d (perhaps 500 or 1000) which display some amount of the desired properties, which are narrowed to even fewer xe2x80x9cleadsxe2x80x9d (perhaps 50 or 100) which are more efficacious. At this point, typically, the lead compounds are assayed for their ADME/PK (absorption, distribution, metabolism, elimination/pharmokinetic) properties. They are tested using biochemical assays such as Human Serum Albumin binding, chemical assays such as pKA and solubility testing, and in vitro biological assays such as metabolism by endoplasmic reticulum fractions of human liver, in order to estimate their actual in vivo ADME/PK properties. Most of these compounds are discarded because of unacceptable ADME/PK properties.
In addition, even optimized leads that have passed these tests and are submitted for FDA clinical trials as investigational new drugs (INDs) will sometimes show undesirable ADME/PK properties when actually tested in animals and humans. Abandonment or redesign of optimized leads at this stage is extremely costly, since FDA trials require formulation, manufacturing and extensive testing of the compounds.
The development of compounds with unacceptable ADME/PK properties thus contributes greatly to the overall cost of drug development. If there was a process by which compounds could be discarded or redesigned at an earlier stage of development (the earlier the better), then great savings in terms of money and time could be achieved. The current art essentially offers no comprehensive method by which this can be done.
A large portion of all drug metabolism in humans and most all organisms is carried out by the cytochrome p450 enzymes. The cytochrome p450 enzymes (CYP) are a superfamily of heme-containing enzymes that include more than 700 individual isozymes that exist in plant, bacterial and animal species. Nelson et al. Pharmacogenetics 1996 6, 1-42. They are monooxygenase enzymes. Wislocki et al., in Enzymatic Basis of Detoxification (Jakoby, Ed.), 135-83, Academic Press, New York, 1980. Although humans share the same several CYP isozymes, these isozymes can vary slightly between individuals (alleles) and the isozyme profile of individuals, in terms of the amount of each isozyme that is present, also varies to some degree.
It is estimated that in humans, 50% of all drugs are metabolized partly by the p450 enzymes, and 30% of drugs are metabolized primarily by these enzymes. The most important CYP enzymes in drug metabolism are the CYP3A4, CYP2D6 and CYP2C9 isozymes. While modeling techniques do exist for predicting substrate metabolism by enzymes other than CYP, no sufficiently accurate technique exists for modeling metabolism by the CYP enzymes. To the extent that modeling techniques are available for other enzymes, they work by analyzing the either the interactions between enzyme and substrate, or the common characteristics for a series of substrates. See, for example, Schramm, xe2x80x9cEnzymatic transition states and transition state analog design.xe2x80x9d Annu Rev Biochem 1998; 67: 693-720; Hunter, xe2x80x9cA structure-based approach to drug discovery; crystallography and implications for the development of antiparasite drugs.xe2x80x9d Parasitology 1997; 114 Suppl: S17-29; Gschwend et al, xe2x80x9cMolecular docking towards drug discovery.xe2x80x9d Mol Recognit 1996 Mar-Apr; 9(2): 175-86.
While these modeling techniques are partially effective for some enzymes, they can be ineffective for the CYP enzymes. This is because the CYP enzymes do not have binding specificities in the way that other enzymes do. CYP3A is almost completely nonspecific from a steric perspective, while CYP2D6 and CYP2C9 are only modestly sterically specific. Gross steric and electrostatic properties of a substrate have a secondary effect on their metabolism by the CYP enzymes, at most. Thus modeling techniques in the current art cannot be used to model CYP enzyme metabolism.
In view of the foregoing importance of the CYP enzymes to drug metabolism, a modeling technique for CYP-substrate interaction and metabolism would be highly beneficial. Such a technique would provide researchers with valuable ADME/PK information on compounds at an early stage in the development process.
The present invention addresses this need by providing methods and systems for identifying reactive sites on a substrate molecule, typically a drug, and determining the relative rates of metabolism of those reactive sites by the CYP enzymes. Determining these relative rates is an important factor in determining the absolute rate of metabolism of the individual sites and the substrate molecule as a whole. This information is also a critical factor in determining whether and how the substrate can be redesigned to improve its ADME/PK properties. In this regard, it is particularly important to know how the relative rates compare to the rate of a non-metabolic side reaction (branch pathway) such as water generation and regeneration of the substrate. The systems and method described there can be used in conjunction with the present invention to provide even more comprehensive information on CYP substrate metabolism.
In a preferred embodiment of the invention, one or more substrate molecule compounds (drugs), or one or more classes of such molecule compounds are presented in a standard representation system such as an organic chemistry string of atoms, a two-dimensional structure, a UIPAC standard name, a 3D coordinate map, or any other commonly used representation. If not already in 3D format, the molecules are converted to 3D format using a 3D formatting software tool such as Corina or Concord and then optimized using AM1.
For each molecule, the reactive sites are identified. The reactive sites are then converted to a radical species to predict their activation energies and reaction rates with respect to the last step of the CYP catalytic cycle. The reaction rate for each reactive site is then compared to the reaction rate for the alternate branch of water decoupling, to determine whether the reactive site is labile, moderately labile or stabile compared to water decoupling. The water decoupling reaction rate is determined by isotope effect information. The molecule as a whole is then characterized in terms of the xe2x80x9crelative reaction ratesxe2x80x9d or xe2x80x9crelative ratesxe2x80x9d for these reactive sites. This information is used to determine whether the molecule has the proper ADME/PK properties, whether it can be redesigned to achieve the most desired ADME/PK properties, and which sites should be modified in order to do this. This information can also be used in conjunction with information obtained about other steps in the CYP catalytic cycle.
One aspect of the invention pertains to methods for predicting an effect of a molecular modification on the metabolism rate of the molecule, the method including the operations of predicting or determining a reaction rate at a first site on the molecule, comparing the reaction rate to that of a branch pathway, and characterizing that site based upon this reaction rate. This allows one to model and predict the effect of the molecular modification on the metabolism of the molecule. The molecule is typically metabolized by a CYP enzyme and the branch pathway is typically a water decoupling reaction. The method can be and is typically repeated for multiple sites on a molecule.
These sites or reactive sites are binned into categories based upon their relative reaction rates with respect to the branch pathway, such as labile for sites that react faster than water decoupling, moderately labile for sites that react at about the same rate as water decoupling, and stable for sites that react slower than water decoupling. The water decoupling activation energy and reaction rate is determined through isotope effect unmasking. The reaction rate of an reactive site is typically determined from its radicalized intermediate form, e.g., by hydrogen abstraction for aliphatic carbon reactive sites and methoxy radical addition for aromatic carbon reactive sites.
Another aspect of the invention pertains to methods for characterizing the reactive site of a substrate molecule, the method including the operations of determining an activation energy for the reactive site, calculating a rate constant for the reactive site based upon its activation energy, and comparing the activation energy or rate constant to a branch pathway associated with the enzyme-substrate complex. The substrate is typically metabolized by a CYP enzyme and the branch pathway is typically a water decoupling reaction. The method can be and is typically repeated for multiple sites on a substrate.
These sites or reactive sites are binned into categories based upon their relative reaction rates with respect to the branch pathway, such as labile for sites that react faster than water decoupling, moderately labile for sites that react at about the same rate as water decoupling, and stable for sites that react slower than water decoupling. The water decoupling activation energy and reaction rate is determined through isotope effect unmasking. The reaction rate of an reactive site is typically determined from its radicalized intermediate form, e.g., by hydrogen abstraction for aliphatic carbon reactive sites and methoxy radical addition for aromatic carbon reactive sites.
Another aspect of the invention pertains to methods for predicting an effect of a molecular modification on the metabolism rate of the molecule, the method including the operations of predicting or determining a reaction rate at a first site on the molecule, comparing the reaction rate to that of a branch pathway, and characterizing that site based upon this reaction rate. This allows one to model and predict the effect of the molecular modification on the metabolism of the molecule. The molecule is typically metabolized by a CYP enzyme and the branch pathway is typically a water decoupling reaction. The method can be and is typically repeated for multiple sites on a molecule.
Another aspect of the invention pertains to computer systems for implementing the methods described above. Another aspect of the invention pertains to computer-program products including a machine-readable medium on which is provided program instructions for implementing one or more of the computer systems or methods described above. Any of the computer system user interfaces or methods of the invention may be represented as program instructions that can be provided on such computer-readable media.